Cerberus–Nodal–Lefty–Pitx Signaling Cascade Controls Left–Right Asymmetry in Amphioxus

Cerberus–Nodal–Lefty–Pitx signaling cascade controls left–right asymmetry in amphioxus

Guang Lia,1, Xian Liua,1, Chaofan Xinga, Huayang Zhanga, Sebastian M. Shimeldb,2, and Yiquan Wanga,2

aState Key Laboratory of Cellular Stress Biology, School of Life Sciences, Xiamen University, Xiamen, Fujian 361102, China; and bDepartment of Zoology, University of Oxford, Oxford OX1 3PS, United Kingdom

Edited by Marianne Bronner, California Institute of Technology, Pasadena, CA, and approved February 21, 2017 (received for review December 14, 2016) Many bilaterally symmetrical animals develop genetically pro- Several studies have sought to dissect the evolutionary history of grammed left–right asymmetries. In vertebrates, this process is un- Nodal signaling and its regulation of LR asymmetry. Notably, der the control of Nodal signaling, which is restricted to the left side asymmetric expression of Nodal and Pitx in gastropod mollusc by Nodal antagonists Cerberus and Lefty. Amphioxus, the earliest embryos plays a role in the development of LR asymmetry, in- diverging chordate lineage, has profound left–right asymmetry as cluding the coiling of the shell (5, 6). Asymmetric expression of a larva. We show that Cerberus, Nodal, Lefty, and their target Nodal and/or Pitx has also been reported in some other lopho- transcription factor Pitx are sequentially activated in amphioxus trochozoans, including Pitx in a brachiopod and an annelid and embryos. We then address their function by transcription activa- Nodal in a brachiopod (7, 8). These data can be interpreted to tor-like effector nucleases (TALEN)-based knockout and heat-shock suggest an ancestral role for Nodal and Pitx in regulating bilat- promoter (HSP)-driven overexpression. Knockout of Cerberus leads erian LR asymmetry, however the picture is complicated by data Nodal Lefty Pitx to ectopic right-sided expression of , ,and ,whereas from other lineages. First, in ecdysozoan lineages, Nodal appears Cerberus overexpression of represses their left-sided expression. to have been lost (7, 9), whereas Pitx is generally symmetrically Cerberus Overexpression of Nodal in turn represses and activates expressed (8) and, where studied, does not function in LR asym- Lefty and Pitx ectopically on the right side. We also show Lefty Nodal Pitx Nodal metry (10, 11). Second, some lophotrochozoans do not show represses ,whereas activates . These data combine asymmetric Pitx expression (8, 12, 13). Third, functional studies on

in a model in which Cerberus determines whether the left-sided EVOLUTION protostome Nodal signaling are currently restricted to molluscs gene expression cassette is activated or repressed. These regulatory (5), so it is unclear whether the regulatory connection between steps are essential for normal left–right asymmetry to develop, as Nodal and Pitx is conserved even in those species that show when they are disrupted embryos may instead form two pheno- asymmetric expression. An additional complication is that Cer and typic left sides or two phenotypic right sides. Our study shows Lefty the regulatory cassette controlling left–right asymmetry was in are also absent from many invertebrate bilaterian lineages (9, place in the ancestor of amphioxus and vertebrates. This includes 14). The extracellular inhibition of Nodal by the proteins encoded the Nodal inhibitors Cerberus and Lefty, both of which operate in by these two genes is a critical component of Nodal signaling in feedback loops with Nodal and combine to establish asymmetric vertebrates (4, 15) and is required for both the generation and Nodal Pitx2 Pitx expression. Cerberus and Lefty are missing from most inverte- maintenance of asymmetric and . brate lineages, marking this mechanism as an innovation in the To examine the evolutionary history of Nodal signaling and its lineage leading to modern chordates. regulation, we turned to amphioxus, the basal chordate lineage and one that forms a typical chordate body plan while also de- amphioxus | Nodal | left–right asymmetry | TALEN | embryonic veloping pronounced LR asymmetries (16). During amphioxus development embryogenesis, the mouth opens on the left side of the head,

ilaterians share three primary developmental axes. The an- Significance Bterior–posterior (AP) and dorsal–ventral (DV) axes define bilaterally symmetrical organization. The third axis, orthogonal Some bilaterally symmetrical animals show genetically pro- to these, is known as the medial–lateral or left–right (LR) axis grammed differences between their left and right sides, for and displays mirror-image symmetry. However, many bilaterian example the placement of the heart and viscera in humans and species deviate consistently from true symmetry, raising funda- other vertebrates. We dissect the regulation of this in embryos mental questions of how symmetry is broken and how different of amphioxus, a close relative of vertebrates that develops developmental programs can unfold on the left and right sides of extraordinary asymmetries as a larva. We demonstrate a sys- an organism (1). In vertebrates, this includes asymmetric devel- tem in which asymmetric expression of the signal gene Nodal is opment of the heart and viscera, disruption of which during controlled by positive and negative feedback loops with its embryogenesis causes a range of human disorders (2). own inhibitors. When this system is disrupted, embryos de- Correct LR organization in vertebrates is regulated by a gene velop mirror-image symmetry with two “left” sides or two “ ” cassette in which right-sided Cerberus (Cer) and left-sided Nodal right sides. Comparison with other animals shows how this and Lefty regulate left-sided expression of the Pitx family gene complex regulatory mechanism has evolved by addition of new Pitx2 and hence morphological LR asymmetry (3). Cer expression feedback regulation to a more ancient signaling interaction. on the right of the embryonic node is required to repress Nodal Author contributions: G.L., X.L., S.M.S., and Y.W. designed research; G.L., X.L., C.X., and signaling, which happens by Cer protein binding directly to Nodal H.Z. performed research; G.L., X.L., C.X., H.Z., S.M.S., and Y.W. analyzed data; and G.L., protein. This restricts the ability of Nodal protein to activate the X.L., S.M.S., and Y.W. wrote the paper. expression of the Nodal gene, leading to an up-regulation of Nodal The authors declare no conflict of interest. Lefty on the left of the embryo. expression is also up-regulated by This article is a PNAS Direct Submission. Nodal and, like Cer, acts as an extracellular inhibitor of Nodal. 1 – G.L. and X.L. contributed equally to this work. Coexpression of Nodal and Lefty can act as an activator inhibitor 2To whom correspondence may be addressed. Email: [email protected] or sebastian. system in the sense originally described by Turing (4). Nodal on [email protected]. Pitx2 the left of the embryo activates expression of , which directs This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. the development of left-sided morphology (3). 1073/pnas.1620519114/-/DCSupplemental.

www.pnas.org/cgi/doi/10.1073/pnas.1620519114 PNAS Early Edition | 1of6 Downloaded by guest on October 2, 2021 +/− −/− Cer ,andCer genotypes in the expected ratio (SI Appendix, Fig. S4), and we assessed the expression of Nodal, Lefty,andPitx in +/− these embryos. Although Cer and wild-type embryos main- −/− tained wild-type expression, Cer embryos showed bilateral expression of Nodal, Lefty,andPitx (Fig. 1 A–F). This indicates Cer is necessary to restrict the expression of Nodal, Lefty,andPitx to the left side of amphioxus embryos. To test whether Cer was also sufficient to regulate these genes, we induced overexpression of Cer using a heat-regulated HSP::Cer construct (SI Appendix, Fig. S5). This generated widespread ectopic Cer expression in 100% (31/31) of embryos (Fig. 1H) and abolished left-sided expression of Nodal, Lefty,andPitx in 100% (84/84), 95% (80/88), and 66% J L N Fig. 1. Cer regulates Nodal, Lefty, and Pitx expression in amphioxus. (46/70) of embryos, respectively (Fig. 1 , ,and ). Combined, (A–F) Expression of Nodal, Lefty, and Pitx in Cer wild-type/heterozygous thesedatashowCer is necessary and sufficient for regulating the + + + − − − (Cer / , Cer / ) or homozygous mutant (Cer / ) embryos. (G–N) Expression lateral expression of Nodal, Lefty,andPitx. of Cer, Nodal, Lefty, and Pitx in Control (uninjected but heat-shocked) and Nodal signaling is transduced by Tgfβ receptors of the Alk4/5/7 Cer misexpressed (Hsp70::Cer-injected and heat-shocked) embryos. Numbers family (22). Pharmacological inhibition of Alk4/5/7 in amphioxus at the bottom right of each panel indicate the number of times the phe- has been reported to block left-sided Nodal, Lefty,andPitx ex- notype shown was observed out of the total number of manipulated em- pression (18), and we found the same in our experiments (Fig. 2 B, bryos. (Scale bar, 50 μm.) All embryos are in dorsal view with anterior to left F H as indicated in the sketch at the bottom left. ,and ). However, the Alk4/5/7 receptor may also mediate signaling by other Tgfβ family ligands, including Activin and Tgfβ (23), raising uncertainty as to the ligand involved in its activation while the gill slits, which initiate opening at the ventral midline, later in amphioxus in vivo. Nodal in amphioxus also has an earlier extend up the right side. Other endodermal organs are similarly developmental role (24), precluding its inhibition by the TALEN asymmetric, and somites also show pronounced LR asymmetry. Cer, method as embryos would not develop to the stage where LR Nodal, Lefty,andPitx are asymmetrically expressed in amphioxus, asymmetry can be addressed. We therefore tested the effect of and pharmacological manipulation of the Alk4/5/7 Tgfβ receptor inducible and ectopic Nodal on amphioxus LR asymmetry. disrupts LR development, suggesting a function for Nodal in am- Overexpression of Nodal using an HSP::Nodal construct (Fig. 2 R, phioxus LR asymmetry (17, 18). However, Nodal function has not T,andV and SI Appendix,Fig.S5), or administration of Nodal been directly tested, and it is not known if Pitx regulates LR protein to developing embryos (Fig. 2 J, N,andP), induced ectopic asymmetric morphology. The functions of Cer and Lefty are also untested, so it is not clear if and how they regulate Nodal signaling. To test these regulatory interactions in amphioxus, we developed transcription activator-like effector nucleases (TALEN)-based methods of removing gene function and heat-shock promoter (HSP)-driven methods of overexpression and deployed them to address the function of all four genes. We show that right-sided Cer represses Nodal expression and find that when Cer function is impaired Nodal becomes bilateral. Nodal activates its own expression and also that of Lefty. Lefty feeds back to repress Nodal, restricting its expression to the left side. This results in left-sided activation of Pitx. We further show that these regulatory steps are essential for normal LR asymmetry to develop. The knockdown or overexpression of Cer, Nodal, Lefty,orPitx results in embryos with either two phenotypic left sides or two phenotypic right sides, with bilateral duplication of most normally lateralized structures in each case. These data show the full regulatory cassette, with both inhibitors acting to restrict Nodal expression to the left side, is present in amphioxus and controls LR morphology. We conclude this pathway represents an ancient chordate character, with the full antagonist-based regulation of Nodal already present in the common ancestor of the chordates. Results Cer, Nodal, Lefty, and Pitx are known to be asymmetrically – Fig. 2. Nodal regulates Cer, Nodal, Lefty,andPitx expression in amphioxus. expressed in amphioxus embryos (18 21), however the relative (A–H) Expression patterns of Nodal, Cer, Lefty and Pitx in control (DMSO) and timing of their expression has yet to be elucidated. We therefore Nodal pathway inhibitor (SB505124)-treated embryos. Embryos were treated examined whether these genes are spatiotemporally activated in from the midgastrula stage until they were collected for gene expression patterns consistent with a regulatory hierarchy. Staged in situ analysis (the 3-somite stage for Cer and around the 8-somite stage for other hybridization demonstrated that right-sided Cer preceded lateral genes). (I–P) Expression patterns of Nodal, Cer, Lefty,andPitx in Control (un- restriction of Nodal, Lefty, and Pitx (SI Appendix, Fig. S1). Fur- treated) and Nodal protein-treated embryos. Embryos were cultured with μ thermore, lateralized expression of Lefty and Pitx preceded that 8 g/mL of mouse recombinant Nodal protein for the same times as above. – of Nodal (SI Appendix, Fig. S1). (Q V) Expression patterns of Nodal, Lefty,andPitx in control (uninjected but heat-shocked) and Nodal misexpressed (Hsp70::Nodal-injected and heat- These temporal expression patterns are consistent with Cer shocked) embryos. Numbers at the bottom right of each panel indicate the regulating the lateralization of the other genes. To test this, we number of times the phenotype shown was observed, out of the total number generated a TALEN knockout heterozygous Cer line (SI Ap- of embryos examined. (Scale bar, 50 μm.) All images are dorsal views with +/− +/+ pendix, Figs. S2 and S3). Crossing Cer animals yielded Cer , anterior to left as indicated in the sketch at the bottom right.

2of6 | www.pnas.org/cgi/doi/10.1073/pnas.1620519114 Li et al. Downloaded by guest on October 2, 2021 right-sided expression of Nodal, Lefty,andPitx. We also found that Lefty and Pitx (Fig. 2 N, P, T,andV), this probably in turn leads to Nodal manipulation fed back on Cer expression, with Alk4/5/7 in- the ectopic expression of Lefty and Pitx seen in Lefty TALEN- hibition inducing ectopic left-sided Cer (as previously reported in injected embryos (Fig. 3 F and H). ref. 18) and Nodal overexpression inhibiting Cer expression in the Pitx homeobox genes are conserved targets of Nodal signaling first somites (Fig. 2 D and L). These results confirm previous in- in vertebrates (3), echinoderms (26), and probably also molluscs ference of the necessity of Nodal for lateralised Pitx expression in (5). In vertebrates, asymmetric Pitx2 is required for correct LR amphioxus, demonstrate that it is also sufficient, and further show morphogenesis, but studies suggest that Pitx2 does not feed back that Nodal also negatively regulates Cer expression. Because we to regulate Nodal expression (27). Although the experiments have shown that Cer inhibits Nodal expression, this suggests a described above show that Cer, Lefty, and Nodal can regulate negative feedback loop in which Nodal represses the expression of Pitx expression in amphioxus, they do not establish whether Pitx its own inhibitor. might also regulate their expression. To test this, we sought to In vertebrate embryos, Lefty also acts as an extracellular in- up- and down-regulate Pitx expression, while monitoring its im- Nodal Lefty Pitx hibitor of Nodal protein, and experiments in an echinoderm pact on the expression of and . Up-regulation of suggest this might also be the case in this lineage (25). To test the by mRNA injection caused significant disruption to early de- function of Lefty in amphioxus, we generated embryos in which velopment, although many of the embryos that did survive the Lefty gene was targeted by injection of a TALEN mRNA (SI through to the neurula stage and beyond displayed ectopic right- Nodal Lefty Appendix,Fig.S6). In 82.4% (98/118) of injected embryos, Nodal sided expression of (45.3%: 24/53) and (20.5%: 9/44) B’ D’ expression became bilateral (Fig. 3D). Furthermore, Pitx and (Fig. 3 and ). To abrogate this early developmental impact Lefty of Pitx mRNA injection, we injected an inducible HSP::Pitx itself also became bilateral, in 49.3% (102/207) and 36.7% SI Appendix HSP::Pitx (40/109) of embryos, respectively (Fig. 3 H and F). To test the construct ( , Fig. S5). In heat-induced -injected embryos, 16.5% (19/115) showed ectopic right-sided sufficiency of Lefty to inhibit normal left-sided gene expression, Nodal H’ Lefty we also injected embryos with an inducible HSP::lefty construct (SI expression (Fig. 3 ). None showed ectopic expression (Fig. 3J’). To block Pitx function, we designed a fusion Appendix,Fig.S5). In the majority of heat-shocked embryos, left- construct linking amphioxus Pitx DNA-binding domain to the sided expression of Nodal and Pitx was lost (Fig. 3 L and N). These Engrailed Repressor (EnR) domain (SI Appendix, Fig. S5) and data demonstrate that, in amphioxus, Lefty can act to inhibit injected the mRNA encoding this into amphioxus embryos. As Nodal expression. In the absence of Lefty, Nodal is ectopically with Pitx mRNA injection, many such embryos developed with D EVOLUTION expressed on the right-hand side (Fig. 3 ). As Nodal up-regulates significant abnormalities before the neurula stage, precluding their analysis for LR defects. However, in embryos that reached the neurula stage and displayed approximately normal AP and DV axial organization, ectopic activation of both Lefty and Nodal was observed on the right side in 71.4% (5/7) and 42.9% (6/14) of cases, respectively (Fig. 3 L’ and N’). In some cases, LR asymmetry phenotypes derived from manipulating gene expression early in development might be explained by disturbed AP/DV organization, which in vertebrates may affect the midline and hence increase the incidence of laterality defects (28). Although this consideration might apply to embryos injected with Pitx mRNA or Pitx–EnR mRNA, which would be translated very early in development, the HSP::Pitx construct was only activated by heat shock after the midgastrula stage, and this also induced ectopic right-sided Nodal expression (Fig. 3H’). These data suggest that, unlike vertebrates, in amphioxus Pitx feeds back to regulate Nodal signaling. The experiments described above show that Cer represses Nodal, that Nodal represses Cer and activates Lefty and Pitx, that Lefty represses Nodal, and that Pitx activates Nodal. An assumption is that left-sided Pitx will drive the development of left- sided morphology. To test this, we allowed embryos in which Cer, Nodal, Lefty,orPitx expression had been manipulated to grow to the late embryonic or larval stages, so we could monitor the impact on the development of LR asymmetric characters. Amphioxus larvae are extremely asymmetric, with the mouth positioned on the left side of the head and the gill slits, which Fig. 3. Lefty inhibits Nodal, Lefty, and Pitx expression, and Pitx over- initiate opening at the ventral midline, expanding to spread up expression or knockdown induces bilateral expression of Nodal and Lefty. the right side. Several internal systems are also distinctly orga- (A–H) Expression patterns of Cer, Nodal, Lefty, and Pitx in control (unin- nized across the LR axis of the head and pharynx, including the jected) and Lefty TALEN mRNA-injected embryos. (I–N) Expression patterns club-shaped gland, preoral pit, and endostyle (Fig. 4 A–C). A suit of Nodal, Lefty, and Pitx in control (uninjected but heat-shocked) and Lefty of asymmetrically expressed marker genes (18) including Krox, misexpressed (Hsp70::Lefty-injected and heat-shocked) embryos. (A’–D’)Ex- FoxE4, Nkx2.1, Pit, and Dkk1/2/4 mark the LR displacement of pression patterns of Nodal and Lefty in control (uninjected) and Pitx mRNA- these structures (Fig. 4 A’, E’, I’, M’, and Q’). To determine the injected embryos. (E’–J’) Expression patterns of Nodal, Lefty, and Pitx in impact of manipulating Cer expression on LR morphology, we control (uninjected but heat-shocked) and Pitx misexpressed (Hsp70::Pitx- allowed manipulated embryos to grow to the larval stage before injected and heat-shocked) embryos. (K’–N’) Expression patterns of Nodal Cer−/− and Lefty in control (uninjected) and Pitx–En mRNA-injected embryos. analyzing morphology and LR marker gene expression. Numbers at the bottom right of each panel indicate the number of times the embryos develop a consistent two-left-side (2-left) phenotype, phenotype shown was observed, out of the total number of embryos ex- with a mouth opening on each side, duplication of the left-sided amined. (Scale bar, 50 μm.) All images are dorsal views with anterior to left structures such as the preoral pit and proximal club-shaped gland, as indicated in the sketch at the bottom right. and loss of the right-sided distal club-shaped gland (Fig. 4 D–F and

Li et al. PNAS Early Edition | 3of6 Downloaded by guest on October 2, 2021 also affected: The first slit appeared to initiate at the ventral −/− midline and like the Cer phenotype failed to extend up either −/− side, however unlike Cer embryos, it also failed to properly open. LR marker gene expression precisely matched this morphology, confirming the duplication and loss of reciprocal structures in the 2-left and 2-right phenotypes (Fig. 4 A’–T’). We also allowed embryos in which Nodal or Lefty had been up- or down- regulated to grow to the larval stage. These manipulations also produced embryos with asymmetry defects, with Nodal overexpression and Lefty knockdown resulting in 2-left phenotypes and Nodal knockdown or Lefty overexpression resulting in 2-right phenotypes (SI Appendix, Fig. S7). As discussed above, injection of Pitx mRNA caused early developmental malformations, however a small number of such embryos did pass through to the larval stage and among them 6.1% exhibited 2-left phenotypes. Likewise, Pitx overexpression via the HSP::Pitx construct, which did not affect early development, also produced 2-left phenotypes in 10.2% of cases. No 2-right phenotypes were observed in either manipulation. Embryos injected with Pitx–EnR were severely malformed by the larval stage, and few pharyngeal structures could be discerned. However, in 7% of cases, paired bilateral mouths were observed, approximating the 2-left phenotype. This is consistent with the ectopic right-sided activation of Nodal in Pitx–EnR-injected embryos (Fig. 3 L’ and N’). These data indicate that the asymmetric Fig. 4. Cer mutation or misexpression affects development of amphioxus LR – – – asymmetry and the expression of organ-specific marker genes. (A–C)Asym- expression of the Cer Nodal Lefty Pitx cassette regulates the LR + − metrical morphologies of control (wild-type, Cer / ,orHsp70::Cer-uninjected morphology of the larva in a predictable way: Any side on which but heat-hocked) larvae. (A) Left lateral view of the pharyngeal region focused Nodal–Pitx are activated becomes phenotypically left. If they are on the right side. (C) Transverse section of larva from A (arrow). (D–F)Sym- not activated, it becomes phenotypically right. metrical morphologies of Cer mutant larvae. (D) Left lateral view of the pharyngeal region focused on the sagittal plane. (E) Dorsal view of the pha- Discussion ryngeal region focused on the ventral side. (F) Transverse section of larva from This study shows that the Cer–Nodal–Lefty–Pitx cassette func- – D (arrow). (G I) Symmetrical morphologies of Cer misexpressed larvae. (G)Left tions in amphioxus to regulate LR asymmetry. Our data allow us lateral view of the pharyngeal region focused on the sagittal plane. (H) Dorsal to construct a regulatory model for the left and right side of view of the pharyngeal region focused on the ventral side. (I) Transverse section of larva from G (arrow). (A’–D’)ExpressionofDkk1/2/4 in the region amphioxus embryos (Fig. 5). The model suggests competitive destined to develop into the mouth opening (arrows). The expression domain feedback on the left side of the amphioxus embryo, with Nodal + + + − isdetectedontheleftsideofCer / or Cer / siblings or control embryos and repressing the expression of its upstream repressor (Cer), while − − on both sides of Cer / mutant embryos but is lost in Cer misexpressed em- activating the expression of its downstream repressor (Lefty) and bryos. (E’–H’) Expression of Pit in the prospective preoral pit (arrows). Pit activator (Pitx). In vertebrates, both Cer and Lefty repress Nodal + + + − transcripts are detected on the left side of Cer / or Cer / siblings or control signaling by directly binding to Nodal protein, whereas Nodal up- −/− embryos and on both sides of Cer mutants but are undetectable in Cer regulates its own expression and that of Lefty (4, 15). Thus, in ’– ’ misexpressed embryos. (I L ) Expression of Nkx2.1 in the prospective endo- both lineages, the establishment of LR identify is dependent on style. Expression of Nkx2.1 in the endostyle can be divided into the left-side the appropriate regulation of Nodal via positive and negative part (arrows) and the right-side part (tandem arrows) along the sagittal plane (indicated by red dotted lines). Although its expression in the left side is du- feedback mediated by extracellular factors. Their focus is − − Pitx plicated in Cer / mutants and lost in Cer misexpressed embryos, its expression establishing asymmetric as the downstream effector, and − − in the right side is lost in Cer / mutants and duplicated in Cer misexpressed disruption of this process in amphioxus or vertebrates leads to embryos. (M’–P’) Expression of FoxE4 in the prospective csg. The csg comprises embryos with disrupted LR asymmetry. The morphological two parts: the left-sided duct (arrows) and the right-sided glandular region outcome in amphioxus is predictable based on the gene expres- − − (tandem arrows). FoxE4 expression in the duct is duplicated in Cer / mutants sion; essentially, if Pitx expression is activated on a side of the and lost in Cer misexpressed embryos, but its expression in the glandular re- embryo, it will develop left-sided morphology, whereas if Pitx is −/− ’– ’ gion is duplicated in Cer misexpressed embryos and lost in Cer mutants. (Q T ) absent, right-sided morphology will develop. Manipulations that Expression of Krox in the duct of the prospective csg (arrows). Its expression is −/− yield bilateral Pitx distributions generate unviable embryos with lost in Cer mutants but duplicated in Cer misexpressed embryos. Embryos in – – A’–T’ areindorsalviewwithanteriortoleft.OnA’, L marks the left side and R 2 left- or 2 right-sided organization, with duplication of the re- the right side, and this applies to all panels from A’ to T’. Numbers at the bottom spective pharyngeal and endodermal structures including the right of each panel indicate the number of times the phenotype shown was mouth, endostyle, and club-shaped gland. These morphologies observed, out of the total number of embryos examined. In some images, red show that, once handedness is established in amphioxus, the two dotted lines separate the left and right of the embryos along the midline. sides develop independently and autonomously according to Genotypes as depicted in Fig. 1. Asterisks in C and F denote the external opening their gene expression and are not noticeably influenced by what of the csg. csg, club-shaped gland; csg-g, right-sided glandular region of csg; en, is happening on the other side of the embryo. endostyle; fgs, first gill slit; m, mouth; op, oral papillae; pp, preoral pit. (Scale bar, – – – μ ’ ’– ’ Although the core Cer Nodal Lefty Pitx pathway appears 50 m.) The scale bar in A also applies to B T . conserved between amphioxus and vertebrates, we also note a difference: In amphioxus, Pitx feeds back on the regulation of Nodal SI Appendix expression. Our model (Fig. 5) suggests that in amphioxus ,Fig.S7). The gill slits, while still initiating and opening the propagation of “leftness” downstream of asymmetric Nodal at the ventral midline, were also affected as they failed to extend up may therefore depend on competition between positive feedback the right side (Fig. 4 D–F and SI Appendix,Fig.S7). Conversely, from Pitx and negative feedback from Lefty. This regulatory link is HSP::Cer larvae developed the reciprocal phenotype, with two– not seen in vertebrates or echinoderms (where Nodal regulation of right-side (2-right) and concomitant duplication of most right-sided Pitx expression has been demonstrated) (26), suggesting it may be structures (Fig. 4 G–I and SI Appendix,Fig.S7). The gill slits were an amphioxus novelty.

4of6 | www.pnas.org/cgi/doi/10.1073/pnas.1620519114 Li et al. Downloaded by guest on October 2, 2021 localized to the gastrocoel roof (31). Whatever the symmetry- breaking mechanism, our data suggest it might work in two ways. Symmetric Cer precedes asymmetric right-sided Cer.Thisin turn precedes asymmetric Nodal expression, but asymmetric Lefty and Pitx expression are detectable before asymmetric Nodal expression yet are regulated by Nodal. Thus, the symmetry-breaking mechanism might act to repress Cer expression on the left, allowing Nodal protein to activate Lefty and Pitx. Nodal autor- egulates, explaining the lag in asymmetric Nodal expression comparted to Lefty and Pitx. Under this model, Nodal negative feedback on Cer would act to lock in this state. We do not know precisely how Nodal feeds back on Cer, although inhibition of the Alk4/5/7 receptor shows it works through this pathway. The second possibility is that, as Nodal feeds back negatively on Cer, the symmetry-breaking mechanism might act to liberate Fig. 5. A gene regulatory network model for the control of LR asymmetry in Nodal from Cer repression. Because Cer represses Nodal by amphioxus. At the gastrula stage, bilateral expression of Cer, Nodal,andLefty direct protein–protein binding (15), this could be via some ad- are observed, but Pitx is not expressed, indicating Nodal regulation of Pitx is ditional extracellular factor preventing or disrupting this in- blocked. During the late gastrula and early neurula stages, Cer expression is teraction. Under this model, Nodal would then repress Cer and maintained on the right side of the embryo. Cer continues to represses Nodal regulate Lefty and Pitx as above. Although compatible with much expression, and as a consequence, downstream activation of Nodal and Lefty is of our data, this does not easily account for the early onset of lost, and Pitx is not activated. In the absence of Pitx, right-sided morphology Cer Lefty Pitx develops. On the left of the embryo, Cer expression is lost during the late asymmetric compared with and . It is also not how gastrula to early neurula stage. Nodal is therefore not inhibited and can ac- the system is thought to work in vertebrates (Fig. 5), where there is tivate the expression of itself, Pitx,andLefty.Left-sidedPitx expression results, so far no evidence for Nodal feeding back on Cer.Wehence and hence left-sided morphology develops. An equivalent diagram summa- consider it a less parsimonious explanation. rizing the interactions in vertebrates is shown for comparison. Our study shows that full complexity of the Cer–Nodal–Lefty–

Pitx cassette was established by the common ancestor of the EVOLUTION chordates and inherited by their vertebrate descendants. Pitx and There are also some aspects of amphioxus morphology on Nodal are also involved in LR specification in molluscs (5) and which our work sheds light. The amphioxus mouth is unusual in may be asymmetrically expressed in some other lophotrochozoans that it opens on the left side of the head, rather than the midline (7, 8), leading to speculation that the role of Nodal signaling in LR as in most animal lineages, including vertebrates. This has led to development was acquired early in animal evolution. However, the speculation that the amphioxus mouth is a derived structure, roles of Cer and Lefty may be more recent innovations. Cer is a possibly evolving from the equivalent of a left-sided coelomic member of the larger Cer/DAN/Gremlin family of Tgfβ signaling pore and not homologous to mouths in other chordates (17). Our Cer – – – regulators. Clear genes have only been identified in chordates, demonstration of its dependence on the Cer Nodal Lefty Pitx although one study suggests they may be older and lost by many pathway supports this theory, as the mouth is regulated as a left- lineages (9). Lefty is a divergent member of the Tgfβ family. It is sided rather than a midline structure. so far found only in chordates and ambulacrarians and in the latter Amphioxus gill slits are also unusual. The first gill slit openings, does function in LR asymmetry (9, 25). Therefore, it is unlikely known as the primary gill slits, appear first at the ventral midline that Cer and Lefty are more broadly involved in LR asymmetry and then extend only up the right side of the head (29). Through across protostomes, and we suggest their incorporations into larval development, gill slits are hence right-sided only. However, regulation of LR asymmetry are innovations of chordates and at metamorphosis, another set of gill slit openings appear, known deuterostomes, respectively, with negative feedback providing as the secondary gill slits. These initiate also on the right side of more nuanced control of Nodal signaling in these lineages. the larva, dorsal to the primary slits. As these secondary slits extend, the primary slits move ventrally and eventually come to lie Materials and Methods on the left side of the head, such that the final adult morphology Animal and Embryo Cultivation and Embryo Microinjection. Amphioxus (Bran- has symmetrical gill slits. This has led to an assumption, dating chiostoma floridae) were originally acquired from Jr-Kai Yu, Institute of Cellular from the 1800s, that the slits that lie on the right side of the larval and Organismic Biology, Academia Sinica, Taipei, Taiwan, and the colony was head are in fact homologous to the left-sided slits in other deu- maintained under previously described conditions (34, 35). Gametes were terostomes (30). In our experiments, the primary slits initiate but obtained using the thermal-shock method (from 20 °C to 26 °C) (34). Fertilization fail to extend in 2-left embryos or to open at all in 2-right embryos. and subsequent culturing of the embryos were carried out essentially as de- – That they initiate is consistent with their positioning at the ventral scribed previously (36) at 27 28 °C unless otherwise stated. Injection solution was prepared containing 20% (vol/vol) glycerol, 5 mg/mL Texas Red dextran (Life midline, suggesting this does not require LR axial information. Technology Co.), and 0.25 μg/μL plasmid DNA or 0.15–1.5 μg/μL synthesized Failure to extend in either sort of embryo suggests subsequent mRNA. Embryo microinjection was performed as previously described (36). morphogenesis requires both left- and right-sided information. Furthermore, that the slits develop further in 2-left embryos than Larval Cultivation and Detection of Cer Mutations in Founder and F1 Amphioxus. in 2-right embryos is in keeping with the historical view that the Cer TALEN mRNA-injected embryos and larvae were reared at 24 °C and fed with primary slits are, primitively, left-sided structures. unicellular algae Dicrateria zhanjiangensis, and juveniles and adults were reared We do not know what breaks symmetry in amphioxus. Studies in a same way as a previous description (34, 35). Each F0 founder was crossed in some vertebrates, in the urochordate Halocynthia, and in with a wild-type animal, respectively. About 30–50 embryos from each cross echinoderms suggest a role for cilia in symmetry breaking (31–33). were collected and lysed with Animal Tissue Direct PCR Kit (Foregene Co.) for mutation efficiency and mutation site analysis (34). Progeny of crosses carrying Although the amphioxus embryo is heavily ciliated at the stages at frame-shift mutations were maintained. F1 genotypes were determined in a which the model suggests symmetry is broken, as yet there is no similar way as described for F0 by cutting a tiny tip of the tail of each animal. The evidence for a functional role for cilia in amphioxus. We consider F1 animals carrying identical frame-shift mutations were maintained and inter- the timing of expression and regulatory interactions consistent crossed to generate F2 generation. The F2 embryos were fixed at different de- with the proposal by Blum et al. for a cilia-based mechanism velopmental stages with 4% (wt/vol) PFA–Mops–EGTAforinsituhybridization

Li et al. PNAS Early Edition | 5of6 Downloaded by guest on October 2, 2021 analysis or morphological examination. Animal husbandry and mutation de- Heat-Shock Experiment. Embryos injected with Hsp70::Cer, Hsp70::Nodal, tection are described in detail in SI Appendix, SI Materials and Methods. Hsp70::Lefty,orHsp70::Pitx DNA plasmids were raised at 25 °C, and heat- shocked at 34 °C in a water bath for 30 min at the early gastrula stage. Plasmid Construct Preparation. The full length of coding sequences of Cer, Uninjected embryos at the same stage were also heat-shocked and used as Nodal, Lefty,andPitx genes was amplified from a gastrula cDNA library and controls. After heat shock, the embryos were returned to 25 °C and allowed then ligated into a pGEM-T-Easy vector (Promega Co.) separately. Further, they to develop until they were fixed with 4% (wt/vol) PFA–Mops–EGTA at de- were recombined into expression vectors pXT7 or P1. The P1 is a construct ini- sired stages for in situ or morphological analysis. tially derived from vector pAcGFP1-1 (Clontech Co.) by integrating a 790-bp regulatory region of B. belcheri Hsp70 gene upstream of the GFP coding se- SB505124 and Nodal Protein Treatment and in Situ Hybridization. Embryos quence (37). A Pitx–EN-dominant negative construct was made by fusing the were treated with 50 μM SB505124 (Sigma Co.) or 8 μg/mL recombinant Pitx DNA-binding domain and the Drosophila EnR domain in the pXT7 vector. mouse Nodal protein (R&D Co.) in four-well dishes [coated with 1.5% (wt/vol) TALENs targeting Cer and Lefty genes were designed and assembled as previously agarose] from early gastrula, and then were fixed at required stages for in situ described (38). TALEN binding sites and primer sequences used for cloning and hybridization or cultured continuously for morphological observation. The – TALEN efficiency assay are shown in SI Appendix,TablesS1 S5. The details for the experiments of in situ hybridization were performed essentially according to a preparation are described in SI Appendix, SI Materials and Methods. previous description (39). All details are described in SI Appendix, SI Materials and Methods. In Vitro Synthesized mRNA and Plasmid DNA Preparation. All plasmid DNAs were prepared using Plasmid Mini Kit (Omega Co.) and linearized with either ACKNOWLEDGMENTS. We thank Dr. Jr-Kai Yu for providing B. floridae an- BamHI (for pXT7-derived constructs) or SacI (for TALEN constructs), extracted imals, and Dr. Dominic Norris for comments on the manuscript. This work by phenol-chloroform, and dissolved in RNase-free water. In vitro mRNA was supported by National Natural Science Foundation of China Grants synthesis was conducted using either T7 (for pXT7-derived constructs) or T3 31471986, 31372188, and 31672246 and Fundamental Research Funds for (for TALEN constructs) mMESSAGE mMACHINE kit (Ambion Co.). the Central Universities of China Grant 20720160056.

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